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Flashcards in Lecture 10 Deck (41)
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1

What are the main distinguishing features of the growth cone

Filopodia – long projections lamellae – web-like fanning between projections

2

What is the difference in actin arrangement in the structures of the growth cone

F-actin is bundled together in a polarised fashion in filopodium whereas in the lamellae they are cross-linked to form a net

3

Describe the different structures of the central peripheral and transitional domains of the growth cone

The central zone or base is a microtubule rich domain. The regional furthest from central domain is the F-actin rich peripheral domain that possesses the filopodia. The transitional zone in the middle of central and peripheral domains

4

Explain the actin treadmilling that is seen in the resting growth cone

F-actin subunits are added at the peripheral zone move through the microfilament and are removed at the central zone. Tubulin is sporadically dragged into the filopodia

5

Growth cones can turn T or F

F – they don’t turn they reorganise

6

What happens when the growth cone comes into contact with an attractive cue

F-actin treadmilling slows down and F-actin begins to accumulate which stabilises the filopodia. A molecular clutch engages the extension over the substrate and an actin-tubulin link pulls the microtubules into the wake of the extending filopodium

7

When a growth promoting cue is encountered two key components lead to filopodial extension what are these

A molecular clutch is engaged and rearward actin treadmilling slows down. Next an actin-tubulin links pulls the microtubules into the wake of the extending filopodium

8

Filopodia are not necessarily attached to the substrate but the central domain is T or F

T

9

Attachment of the growth cone to a substrate is enough to drive forward movement T or F

F – need a stimulus or a cue to trigger rearrangement of the cytoskeleton

10

Describe the evidence for a molecular clutch in growth cone extension

Micropatterned substrate containing dots of N-cadherin was generated. Neurons were then transfected with an N-cadherin-GFP fusion protein and allowed to move over the substrate. N-cadherin in the cells will bind homophilically to the N-cadherin dots on the substrate. It was observed the following extension of the growth cone the labelled GFP-N-cadherin fusion proteins become localised to the regions of the substrate where the N-cadherin dots were indicating a link between the extracellular environment and the cytoskeleton

11

Describe the effects of actin cross linking on engagement of the molecular clutch

The clutch could also be controlled by actin cross-linking. Uncrosslinked F-actin has little strength with no net movement resulting in treadmilling. In contrast branched and crosslinked F-actin has strength to push membrane forward

12

Which family of GTPases are responsible for the coordination of actin cytoskeletal organisation and the control of neuronal morphology movement and polarity

Rho GTPases

13

What two factors are important in the regulation of GTPases

GAPs and GEFs

14

In an inactive state GTPases are bound to GDP what is required to activate signalling

Displacement of the GDP by GTP activates the GTPase and initiates signalling

15

What is the result of the intrinsic nature of GTPases to hydrolyse GTP

Hydrolysis of the bound GTP by the GTPase releases a phosphate and switches it back to an inactive state

16

Nucleotide-free GTPases are extremely energetically favourable T or F

F – its extremely unfavourable

17

What are GEFs and what is the role of these proteins in the cyclic nature of GTPase activity

Guanine nucleotide exchange factors stabilise GTPases in a transition state so that GTP can then bind after GDP release

18

GTPase activating proteins are responsible for catalysing the hydrolysis of the GTP bound to GTPases thus do they act as positive or negative regulators of GTPase signalling

GAPs are negative regulators of GTPase signalling as they promote the catalyses of GTP hydrolysis to the inactive GDP-bound form

19

What is the role of guanine nucleotide dissociation inhibitors

GDIs effectively pull the GDP bound GTPases out of the cycle and hold it in the cytoplasm to create a pool of inactive GTPases

20

What changes happen at the molecular level as a result of GTP nucleotide binding to GTPases

This causes a very small conformational change dictated by the presence of a final phosphate that changes the orientation of the switch 1 and switch 2 domains. This leads to an activation of signalling

21

What are the three members of the Rho family of GTPases

RhoA Rac1 and Cdc42

22

What is the role of Cdc42

Cdc42 is a RhoGTPase that controls the polymerisation of actin filaments and the formation of actin spikes or filopodia

23

What is the role of Rac1

Rac1 controls the organisation of new actin filaments particularly branched actin into dynamic ruffling structures or lamellipodia

24

What is the role of RhoA

RhoA stabilises and consolidates actin filaments into a more rigid skeletal framework known as stress fibres

25

Describe a loss of function approach that can be used to elucidate the precise function of GTPases

Create a dominant negative mutant GTPase with a point mutation in the nucleotide-binding site. This will result in a GTPase that is always off and inhibitory due to never binding to GTP. The dominant negative effect of this mutant is due to its binding to and mopping up of active GEFs to prevent their action on functioning GTPases. By binding to these inhibitory mutant GTPases the GEFs are no longer available to activate other functions wild type GTPases.

26

Describe a gain of function approach that can be used to elucidate the precise function of GTPases

Create a constitutively active GTPase mutant that is always on and remains in the GTP-bound form. This will perturb GTP hydrolysis and creates an always active GTPase

27

What is the result of microinjection of constitutively active RhoA into quiescent cells

Leads to the formation of stress fibres

28

What is the results of microinjection of dominant negative RhoA into active cells

Leads to the loss of stress fibres

29

What is the result of microinjection of constitutively active Rac or Cdc42 into cells

Leads to the formation of membrane ruffles or filopodia respectively

30

What is the specific role of Rac Cdc42 and RhoA in the regulation of axon growth

Activated Rac and Cdc42 are positive regulators of axon growth whereas activated RhoA is a negative regulator